Methods of inhibiting phospholipase A2 and phospholipase A2 stimulator activities

ABSTRACT

A method for inhibiting PLA 2  or suppressing the activity of a PLA 2  stimulator in a body fluid of an animal with lung disease involves adding Annexin I, Annexin VIII or a mixture thereof to the body fluid.

RELATED CASES

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/794,941, filed Feb. 4, 1997, now U.S. Pat. No. 5,849,502which is a division of U.S. patent application Ser. No. 08/443,511,filed May 18, 1995, now U.S. Pat. No. 5,658,877.

This invention was made with United States government support awarded bythe following agencies: NIH, Grant No: HL38744; HL46478. The UnitedStates Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention generally relates to annexins. More particularly,it relates to methods of inhibiting phospholipase A₂ (PLA₂) and theactivity of PLA₂ simulators which employ Annexin I and Annexin VIII.

BACKGROUND OF THE INVENTION

Annexins are a group of calcium-dependent, phospholipid-bindingproteins. The calcium and phospholipid binding sites of most annexinsare located in four repeated and highly conserved regions each of whichcontains about 70 amino acids. These proteins are widely distributed andat least nine members of the annexin family of proteins have beenidentified in mammalian tissues.

The lung is rich in annexins. Several members of the annexin family ofproteins with apparent molecular weights ranging from about 32 to about40 kDa have been isolated from lungs of animals. Annexin I, a 36 kDphospholipid binding protein, 36 kDa(PLBP), appears to be the mostabundant of the annexin family of proteins. It is present in the lung ingreater amounts than the related Annexin VIII, a 33 kDaphospholipid-binding protein, also known as 33 kDa(PLBP).

Compared to the whole lung, the alveolar epithelial type II cells inwhich the pulmonary surfactant complex is synthesized, stored andsecreted have higher expression levels of Annexin I. In addition to theintracellular localization of Annexin I in alveolar type II cells, thisprotein has been found in lung lavage fluids from human and animals. Alikely source of the Annexin I in the lung lavage fluid is the alveolartype II cells.

Using an antibody to Annexin I, researchers have found Annexin I and asmaller protein in the bronchoalveolar lavage (BAL) fluid of patientswith lung diseases. The smaller protein was found to be a proteolyticbreakdown product resulting from the action of neutrophil elastase uponAnnexin I in the patients' BAL fluid. The discovery of the Annexin Ibreakdown product in human BAL fluid samples is consistent with thereport that the Annexin I N-terminal region is subject to cleavage byvarious proteases to yield breakdown products.

I have discovered that a breakdown product of Annexin I, which has amolecular weight of about 33 kDa (33 kDa(BP)), immunoreacts withanti-Annexin I antibodies. I also have discovered that 33 kDa(BP) iscytotoxic and that it is present in higher concentrations in the BALfluid of patients with lung diseases, including cystic fibrosis (CF),and premature infants with chronic lung disease, bronchopulmonarydysplasia, (BPD) than in normal humans. In addition, the BAL fluid ofpatients with lung disease was found to contain less Annexin I than thatof normal humans.

Based on these and other discoveries, I have found that administeringAnnexin I or Annexin VIII, to patients with lung disease can bebeneficial to such patients. I also have made the further discovery thatthe administration of Annexin I or Annexin VIII, can be beneficial inthe treatment of endotoxin toxicity and inflammation. The majorpathogenesis of endotoxin toxicity leads to inflammation and septicshock.

Bacteria or bacterial products, such as endotoxin from gram-negativebacteria, activate host responses during infectious and inflammatoryprocesses. Endotoxin, also known as lipopolysaccharide (LPS) for itschemical structure consisting of a polysaccharide part and a hydrophobiclipid part, can induce a wide variety of different types of cellsincluding macrophages, polymorphonuclear leukocytes, and endothelialcells to release a number of inflammatory mediators, such asprostaglandins or cytokines. In localized infections, endotoxin islargely restricted to inflammatory sites, enhancing host defense.However, if the infection is not brought under control, endotoxin and/orinflammatory mediators may reach the circulation, predisposing themicrovasculature to thrombosis and can lead to systemic endotoxemia orsepsis and associated complications including septic shock, adultrespiratory distress syndrome, and multiorgan failure.

Septic or endotoxin shock is an acute and serious cardiovascularcollapse resulting from the systemic response to a bacterial infection.It is manifested by hypotension, a reduced response to vasoconstrictors,generalized tissue damage and multi-organ failure. It is the most commoncause of death in the intensive-care unit; there are about 400,000 casesof septicemia per year in the United States with mortality rates between25% and 50%. The steadily increasing incidence of septic shock stemsfrom an increasing proportion of elderly in the population, increasingfrequency of invasive surgical procedures, extensive use ofimmunosuppressive and chemotherapeutic agents, and increasing prevalenceof chronic debilitating conditions. Because the mechanisms underlyingsepsis and septic shock are not yet known, therapeutic interventionshave been largely ineffective. At present, there is no effectivetreatment for septic shock.

Phospholipase A₂ (PLA₂) is an enzyme which plays a role in a pathwaywhich can lead to inflammation. Inflammation in the lung is caused byinflammatory mediators released by cells, including histamine,cytotoxins (tumor necrosis factor alpha, interleukin-1, etc), andleukotrienes. These mediators cause blood vessels to leak and lungtissues to swell, and act to contract the smooth muscles of the airwaysto cause bronchial constriction. The inflammation response, originallydesigned to combat threats, is in itself the mediator of substantialtissue damage. Patients with conditions such as asthma, cystic fibrosis,and bronchopulmonary dysplasia experience chronic inflammation of thelung.

Phospholipase A₂ is an important enzyme in a synthetic pathway which canlead to the formation of arachidonic acid. Arachidonic acid in turn maybe acted upon by cyclooxygenase to form prostaglandins, by lipooxygenaseto form leukotrienes, and by monooxygenase to form epoxides all whichcan cause inflammation.

Compounds that interfere with this pathway can be useful in preventinginflammation. For example, prostaglandin inhibitors are widely known andused for inflammation, and a lipooxygenase inhibitor to preventformation of leukotrienes has recently come on the market for treatmentof asthma.

It would be advantageous to have methods of inhibiting PLA₂ activity andsuppressing the activity of PLA₂ simulators, thereby preventing ortreating inflammation, especially that which accompanies lung diseases,including cystic fibrosis (CF) and bronchopulmonary dysplasia (BPD). Italso would be advantageous to have a method of evaluating agents thatmight be effective to suppress the activity of PLA₂ simulators in apatient's body fluid.

SUMMARY OF THE INVENTION

It is an object of the present invention to disclose novelpharmaceutical compositions containing annexins and methods ofinhibiting PLA₂ or suppressing the activity of PLA₂ simulators which canbe used in treating lung inflammation and lung disease in patients andin methods for treating patients with endotoxin shock.

I have discovered a method of inhibiting the effects of PLA₂ andsuppressing the activity of PLA₂ simulators in the body fluid of ananimal which comprises bringing a safe amount of Annexin I, Annexin VIIIor a mixture thereof into contact with an animal fluid containingphospholipase A₂ (PLA₂) or a PLA₂ stimulator which amount is effectiveto inhibit the PLA₂ activity and suppress the activity of the PLA₂simulators in the body fluid.

In a preferred embodiment of the method, the Annexin I or Annexin VIII,or a mixture thereof, is brought into contact with the bronchoaleveolar(BAL) fluid of a patient by inhalation of a pharmaceutical compositioncontaining Annexin I, Annexin VIII or a mixture thereof.

The pharmaceutical compositions of the present invention comprise amember selected from Annexin I, Annexin VIII, and mixtures of thosepolypeptides, in combination with a pharmaceutical diluent(s) and/or anexcipient(s). The preferred compositions also contain a source ofcalcium ions (Ca⁺²). Especially preferred are compositions which aresuitable for instillation into the bronchial system of an animal.

In another embodiment of the method of the invention which is useful indetermining if a candidate anti-inflammatory agent can inhibit thestimulation of the activity of a PLA₂ stimulator in a body fluid, theagent is added to a body fluid known to contain such a stimulator andthe agent's inhibitory activity determined by measuring the amount ofPLA₂ produced.

These and other objects of the invention will be apparent from thedescription and examples herein.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The preferred pharmaceutical compositions are those which contain as theactive ingredient Annexin VIII, 33 kDa(PLBP). The reason that theAnnexin VIII is preferred is that it is as active as Annexin I and it isless likely to be broken down into cytotoxic products by enzymes.

In addition to Annexin VIII, the preferred compositions for instillationinto the airway of an animal will contain a source of calcium ions and asurfactant. The compositions also may contain pharmaceutical diluentsand excipients which are customary for aerosols or other dosage formsfor instillation of drugs into the airways of animals.

In the preferred method of inhibiting the activity of PLA₂ andsuppressing the activity of a PLA₂ stimulator in a body fluid of apatient the preferred composition is instilled directly into thepatient's lungs. In an alternative method, lung fluid is collected froma patient in whom it is desired to inhibit PLA₂ activity or suppress theactivity of a PLA₂ stimulator, the Annexin I, Annexin VIII or a mixturethereof is added to the lung fluid and the lung fluid is returned to thepatient.

The practice of the present invention will be further understood by thedescription of the experimental work that follows.

EXPERIMENTAL WORK I. Degradation of Annexin I in Lung Disease

A study was conducted to determine whether degradation of Annexin Ioccurs in BAL fluid of patients with lung disease, such as cysticfibrosis (CF) and bronchopulmonary dysplasia (BPD). The lung disease ofCF is characterized by bacterial infection and inflammation. As aresult, the patient's mucus contains high amounts of proteases,particularly, the elastase from the neutrophils and from the organismPseudomonas aeruginosa that colonized the respiratory tract of the CFpatients. It was speculated that these proteases may break downproteins, including annexins, in the bronchi and bronchiole. The lungdisease of premature infant BPD is characterized by oxyradical-mediatedacute lung inflammation and injury. These premature infants survivedrespiratory distress syndrome (RDS) after intensive care but sufferedoxygen toxicity and developed bronchopulmonary dysplasia, a chronic lungdisease. The BAL fluid of these patients also contained a high level ofproteases derived from neutrophils in addition to the oxyradicalsubstances. In this study, rabbit lung Annexin I, which is equivalent tohuman Annexin I, was used and specific antibodies were raised againstrabbit lung Annexin I to analyze the distribution of Annexin I in BALfluid from CF patients and BPD patients and to determine the changes inAnnexin I structure and functional activity.

Isolation of lung annexins. Rabbit lung Annexin I was isolated from thecytosolic fraction of the lungs from two adult rabbits by knowntechniques. Human lung Annexin I was isolated from post-mortem lungtissue.

Isolation of lung annexins and preparation of anti-Annexin I antiserum.The two rabbit lung calcium-dependent phospholipid-binding proteins,Annexin I or 36 kDa(PLBP) and Annexin VIII or 33 kDa(PLBP), wereisolated from cytosolic fraction of the lungs from adult rabbit by knowntechniques (Tsao FHC, Biochimica et Biophysica Acta. 1045 (1990) 29-39,incorporated by reference.)

Isolation of Lung-soluble Fraction

An adult New Zealand white rabbit was anaesthetized with an intravenoussodium pentobarbital solution (20 mg pentobarbital and 1000 unitsheparin per kg body weight). Tracheostomy was performed and the lung waslavaged with 20 ml saline three times. The trachea was then connected toa respiratory pump (10 ml/stroke, 25 strokes/min). The thorax was openedand the animal was exsanguinated. The lungs were perfused in situ withsaline via the pulmonary artery, removed from the carcass and dissectedfrom nonlung tissue. Lung tissue was minced and homogenized in volumesof 0.01 M Tris-HCl/0.33 M sucrose buffer (pH 7.4) containing 5 mM2-mercaptoethanol and 1 mM EDTA in a Brinkmann Model PT 10/35homogenizer (Brinkmann Instruments, Westbury, N.Y.). In some studieswith buffer, and in buffers used in the protein isolation describedlater, the buffers contained 0.1 mM phenylmethylsulfonyl fluoride andpepstatin A (10 μg/ml) (Sigma) to inhibit any proteolytic activity. Thesoluble (cytosolic) fraction was obtained from the supernatant of105,000×g centrifugation of the post-mitochondria fraction as describedearlier [F. H. C. Tsao, Biochim. Biophys. Acta 575:234-243, 1979]. Theisolated soluble fraction was stored at −20° C. before use.

Isolation of Ca²⁺-dependent Phospholipid-binding Proteins

Lung-soluble fraction obtained from two adult rabbit lungs was titratedto pH 5.5 on ice followed by centrifugation at 4° C. at 6600×g with aBeckman Model L centrifuge (Beckman Instruments, Palo Alto, Calif.) for10 minutes. The pellets were discarded. The supernatant was titrated topH 7.4 followed by the addition of ammonium sulfate crystals with gentlestirring on ice. The final concentration of ammonium sulfate in thesolution was 70%. The precipitated proteins were pelleted bycentrifuging the mixture at 15,000×g at 4° C. for 20 minutes. Thesupernatant was discarded. The pellets were suspended in about 10 ml0.01 M Tris-HCl buffer containing 5 mM 2-mercaptoethanol, 1 mM EDTA and0.15 M NaCl at pH 7.4 (buffer A).

SEPHADEX G-100 Column Chromatography

Half of the protein solution obtained from ammonium sulfateprecipitation was applied to a SEPHADEX G-100 column (2.6×55 cm)preequilibrated with buffer A. The proteins were eluted with 3.5 ml pertube of buffer A at a flow rate of 12 ml per hour. The protein contentof the eluted fractions was estimated from an absorbance at 280 nm witha Gilford Model 252 photometer (Gilford, Oberlin, Ohio) adapted with aBeckman DU monochromator. The activity of PLBPs (phospholipid-bindingproteins) in the eluted fractions was determined by the fusion assaydescribed later. Protein isolation was carried out at 4° C. The secondpart of the ammonium sulfate-precipitated protein solution was runthrough the SEPHADEX G-100 column by the same procedure.

Phenyl Sepharose CL 4B Column Chromatography

The PLBP fractions obtained from the SEPHADEX G-100 column were pooledand ultrafiltered through an Amicon DIAFLO ultrafilter with an UM 100membrane (10,000 molecular weight cutoff) (Amicon, Danvers, Mass.). Thecondensed protein solution in about 2 ml buffer A was applied to aPHENYL SEPHAROSE column (2×6 cm), preequilibrated with buffer A. Thecolumn was first eluted with 50 ml buffer A, then with 100 ml H₂O. Theelution volume of each fraction was 1.8 ml with a flow rate of 12 ml perhour.

Hydrophobic Interaction High-performance Liquid Chromatography

The PLBP fractions collected from the phenyl Sepharose column in bufferA were pooled and condensed by ultrafiltration using an Amicon UM 10membrane and by Amicon CENTRICON™-10 microconcentrators (10,000molecular weight cutoff). The concentration of NaCl in the condensedprotein solution was adjusted to 3 M and the protein solution wasapplied to a SPHEROGEL TSK PHENYL 5PW (10 μm particle size) column(7.5×75 mm) that was assembled onto a Beckman gradient HPLC system. Thecolumn was washed with 60 ml of buffer A containing 3 M NaCl (buffer B)and was then eluted with a linear descending NaCl gradient of buffer Bagainst buffer A. The rate of gradient change was 2% of buffer B/min.for the first 17 minutes and then dropped to 1% of buffer B/min. for theremaining 40 to 50 minutes. The flow rate was 1 ml/min. per fractiontube.

Sometimes, a second run of the PLBP fractions by the HPLC was needed toget higher purity of the proteins if the sample was overloaded in thefirst run. The conditions of the second run were the same as those ofthe first run. The 36 kDa(PLBP) was identified to be rabbit Annexin I(Tsao F. H. C., et al. Biochimica et Biophysica Acta. 1081 (1991)141-150.) Human lung Annexin I was isolated from post-mortem lung tissueby the same techniques. The purified rabbit lung Annexin I was used asantigen to raise specific antibodies in guinea pigs. The guinea pigantiserum to rabbit lung Annexin I (gpAb-Anx-I) was highly specific forrabbit lung Annexin I and cross-reacted with human Annexin I (Tsao F. H.C. et al. Biochimica et Biophysica Acta. 1213 (1994) 91-99.)

Analysis of annexin in bronchoalveolar lavage (BAL) fluid samples.Bronchoalveolar lavage fluid was obtained from normal volunteersubjects, patients with CF, patients with interstitial lung disease(ILD), and patients with bronchopulmonary dysplasia (BPD). The BALfluids were concentrated 5 to 10-fold by centrifugation (3000×g at 4°C.) using Amicon CENTRICON10 filters (molecular weight cut-off of 10kDa) (Beverly, Mass.). Fluid retained by the filter was saved forfurther analysis. An aliquot of BAL fluid sample containing 0.1 mg oftotal proteins was lyophilized in a SPEED VAC (Savant Instruments, Inc.,Farmingdale, N.Y.) to dryness. The sample proteins were resuspended in20 μl of sample buffer containing sodium dodecyl sulfate (SDS) anddenatured in boiling water for 5 min. The proteins were separated by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) under the denaturedconditions using a vertical 10% SDS gel (7×8 cm). Proteins on the SDSgel were then electrophoretically transferred onto a nitrocellulosemembrane. Annexins on the membrane were immunoblotted by the polyclonalantibody raised in guinea pig against rabbit lung Annexin I(gpAb-anx-I). As specified, in some studies proteins on the SDS gel werevisualized by silver staining (Silver Staining Kit, Sigma Chemical Co,St. Louis, Mo.). The isoelectric point (pI) values of annexins in theBAL fluid samples with 50 μg of total proteins were determined byisoelectric focusing (IEF) using an IEF agarose gel with pH rangebetween 3 and 10 (10×12.5 cm, FMC, Rockland, Me.). Proteins on the IEFgel were transferred to a nitrocellulose membrane by capillary force,and annexins on the membrane were analyzed by Western blot usinggpAb-anx-I.

Effects of BAL fluid from CF patients on the activity and structure ofAnnexin I. An amount of purified rabbit lung Annexin I was incubatedwith CF BAL fluid samples in a ratio of 1 μg Annexin I/20 μg BAL proteinin 10 μl of 0.01 M Tris-HCl, pH 7.4 (for Western blot as describedabove), or in a ratio of 10 μg Annexin I/100 μg BAL protein in 50 μl of0.01 M Tris-HCl, pH 7.4 (for Annexin I activity measurement), at 37° C.for 1 h. The Annexin I activity was determined by measuring theaggregation of ¹⁴C-labeled phosphatidylcholine unilamellar liposomes tomultilamellar liposomes by known techniques.

In a separate experiment, an amount of 0.2 mg of purified rabbit lungAnnexin I was incubated with BAL fluid containing 0.42 mg total proteinsin 0.3 ml of 0.01 M Tris-HCl, pH 7.4, at 37° C. for 2 h. After reaction,the reaction mixture was centrifuged at 100,000×g for 10 min. Annexin Iin the supernatant was isolated by HPLC C4 Vydac reverse phase. Thepurity and the molecular weight of Annexin I obtained from HPLC reversephase column were examined by SDS-PAGE and Western blot. The N-terminalsequence of Annexin I from HPLC reverse phase was determined using anautomated Model 477A Liquid Pulse Sequencer and Model 475A Gas PhaseSequencer with on-line Model 120A PTH Analyzer and 610A Data AnalysisSystem (Applied Biosystems, Foster City, Calif.).

Western blot analysis of annexins in human BAL fluid. With the use of100 μg total BAL fluid proteins, Annexin I was detected in BAL fluidsamples from normal volunteers by Western blot. Annexin I in one of the10 normal volunteer BAL samples was only barely detected. Annexin I alsowas present in all 12 BAL fluid samples from patients with interstitiallung diseases. Small amounts of an immunoreacted protein with molecularweight around 33 kDa also was observed in some of the samples. This 33kDa protein did not bind with phospholipid and was determined to be the33 kDa(BP) breakdown product of Annexin I. It appeared that the BALfluid samples of patients with interstitial lung disease which had about20% neutrophil among total BAL fluid cells also contained small amountsof the 33 kDa(BP). In contrast, in 20 BAL fluid samples from CFpatients, 17 samples had no Annexin I. In 11 of the 17 samples with noAnnexin I, the only immunoreactive protein was the 33 kDa(BP). The other6 among the 17 BAL fluid samples had no detectable immunoreactiveproteins at all. Among the 20 CF BAL specimens, only three samples hadAnnexin I, but two of these three samples also contained theimmunoreactive 33 kDa(BP). Interestingly, the three CF BAL samples whichcontained Annexin I also had lower neutrophil elastase activities. Allthe BAL fluids of CF patients contained neutrophils in concentrationsover 10⁵ cells/ml, compared to the very low concentrations ofneutrophils in normal volunteers and patients with interstitial lungdiseases. The additional two BAL fluid samples from normal volunteersalso contained Annexin I, but one of these two samples also containedthe 33 kDa(BP).

Conversion of Annexin I to 33 kDa(BP) by CF BAL fluid and elastase. Theincubation of purified rabbit lung Annexin I (1 μg) with four differentCF BAL fluid samples (20 μg protein) yielded 33 kDa(BP) which wasimmuno-recognized by the antibody. The 33 kDa(BP) was solely derivedfrom substrate of rabbit lung Annexin I since the four CF BAL samplesemployed in the tests contained only 20 μg of total proteins in whichlittle Annexin I and 33 kDa(BP) could be detected. Under the reactionconditions, three BAL fluid samples converted most of the Annexin I tothe 33 kDa(BP), whereas one BAL fluid sample degraded less Annexin I tothe 33 kDa(BP). Interestingly, that CF BAL fluid sample also containedboth endogenous Annexin I and 33 kDa(BP), whereas the other three CF BALsamples used in the reactions had no Annexin I but only the 33 kDa(BP).

The Annexin I breakdown product, the 33 kDa(BP), generated by a CF BALfluid sample had a basic isoelectric point value with pI at 8.5, whichwas markedly different from the pI of 6.0 of Annexin I or the pI of 5.5of 33 kDa(PLBP). The M_(R) values of Annexin I and 33 kDa(PLBP) andtheir structures were determined as described in the literature.

The incubation of purified rabbit lung Annexin I with CF BAL fluidsamples in which Annexin I was absent resulted in a decrease in AnnexinI activity in liposome aggregation. Contrarily, the incubation of rabbitlung Annexin I with a CF BAL sample in which endogenous Annexin I waspresent did not affect Annexin I activity.

Elastase also degraded rabbit lung Annexin I into the 33 kDa(BP) whichwas immunorecognized by gpAb-anx-I. The presence of phenylmethylsulfonyl fluoride (PMSF) in the reaction solution totally inhibited theproteolytic hydrolysis of Annexin I catalyzed by elastase.

In the experimental work described Annexin I was present in all the BALfluid samples from normal volunteers. The finding of Annexin I in BALfluid from normal volunteers was consistent with the previous reportsthat Annexin I was present in lung lavage fluid from animals and humans.Little degradation of Annexin I was observed in the BAL fluid samplesfrom normal volunteers. However, degradation of Annexin I appeared to becommon in all the BAL fluid samples from CF patients. In most of the CFsamples, Annexin I was completely degraded to 33 kDa(BP). Only a few CFsamples contained any Annexin I, but even those samples also had the 33kDa(BP) protein. Although Annexin I was present in all the BAL fluidsamples from patients with interstitial lung diseases, some of thesamples also contained the 33 kDa(BP) protein.

It is interesting to note that among the BAL fluid samples from patientswith interstitial lung diseases, the appearance of 33 kDa(BP) wasassociated closely with relatively higher percentage of neutrophils inthese samples. Since all the BAL fluid samples from CF patientscontained abundant neutrophils, it was concluded that the degradation ofAnnexin I to the 33 kDa(BP) was associated with neutrophils. Likely, thehigher the neutrophil elastase in the BAL fluid, the more degradation ofAnnexin I took place. For those CF BAL fluid samples which had lowelastase activity, Annexin I was present in the BAL fluid. Thus, thebreakdown of Annexin I in BAL fluid was associated closely with thedegree of lung inflammation in the CF patients.

The proteolytic activity in the CF patients' BAL fluid further wasconfirmed by the degradation of purified rabbit lung Annexin I incubatedin reaction mixtures containing CF BAL fluid. The breakdown product 33kDa(BP) of rabbit lung Annexin I catalyzed by CF BAL fluid structurallywas nearly identical to the human lung Annexin I breakdown product inthe CF BAL fluid samples, i.e., same molecular weight and pI between8.5-9.0. The Annexin I breakdown product 33 kDa(BP) protein had a pIvalue which was distinct from Annexin VIII, 33 kDa(PLBP), which was anacidic pI of 5.5. Both rabbit lung Annexin I and Annexin VIII aggregatednegatively charged vesicles in a calcium-dependent manner, an importantannexin functional activity. The degradation of rabbit lung Annexin Icatalyzed by CF BAL fluid markedly reduced the Annexin I functionalactivity, in other words, the Annexin I breakdown product 33 kDa(BP) wasfunctionally inactive in vesicle aggregation.

Although previous studies suggested that the degradation of Annexin I inBAL fluid from patients with lung diseases was due to the elastasehydrolytic activity, we found that the cleavage site of Annexin I was atthe N-terminus Val-36, an elastase substrate specific cleavage site.Also the cleavage site of Annexin I was determined with rabbit lungAnnexin I which was used as the substrate. Both rabbit lung Annexin Iand human lung Annexin I have nearly identical amino acid sequences inthis region. It also was determined that human lung Annexin I could becleaved at Ser-37. This suggested that degradation of Annexin I couldoccur at more than one position at the N-terminus. It has been shownthat the N-terminus of Annexin I can be cleaved at several positions bydifferent proteases, such as cathepsin D, calpain or plasmin, which havebeen demonstrated to cleave human annexin I at Trp-12, Lys-26 or Lys-29,respectively. The N-terminus truncated Annexin I has been shown toeither increase or decrease the binding affinity with calcium andphospholipid, depending on the N-terminus-truncated position. It hasbeen demonstrated that the removal of 36 amino acids at the N-terminusof Annexin I by the proteases in CF patients' BAL fluid diminished theAnnexin I functional activity in vesicle aggregation and fusion,indicating that the N-terminus is required for Annexin I functionalactivity.

The source(s) of Annexin I in BAL fluid are not known with certainty. Ithas been found that alveolar epithelial type II cells are rich inAnnexin I. Though the role of Annexin I in the type II cell is notclear, it might be associated with lung surfactant and possibly secretedby type II cells. The pulmonary surfactant appears not only to beessential to stabilize alveoli from collapse at the lowest volume duringexpiration, it also may play an important role in mucociliary clearancein the respiratory tract. The abnormal surfactant phospholipidcomposition in the mucus of CF patients not only may contribute to theabnormal mucociliary transport, but it also may cause the collapse ofterminal airways. The degradation of Annexin I in the BAL fluid in CFpatients was not only a sensitive indicator of the high levels ofneutrophils and elastase in the inflammatory lung, it also was anindication of the decreased anti-inflammatory activity due to thereduction of the levels of Annexin I in the lungs of these patients.Similarly, Annexin I in the BAL fluid samples from five BPD patients wasall degraded to 33 kDa(BP). These patients had acute lung inflammationand injury.

From the foregoing, it was apparent that administering Annexin I,Annexin VIII, or mixtures thereof to patients could be beneficial tosuch patients.

II. Isolation and Structural Determination of 33 kDa(BP)

The rabbit lung Annexin I, after incubation of Annexin I with CF BALfluid at 37° C. for 2 h, was purified by HPLC reverse phase column. ThisAnnexin I was eluted as a single peak at the 35-min elution time fromthe column. Some minor proteins, presumably from BAL fluid, were elutedearlier than Annexin I. The apparent molecular weight of this Annexin Iwas determined to be 33 kDa by SDS-PAGE. Thus, this annexin protein wasdesignated as Annexin I breakdown protein, or 33 kDa(BP).

Amino acid sequence determination of 33 kDa(BP) and human lung AnnexinI. The 15 amino acid residues determined for the sequence of the 33kDa(BP) protein derived from rabbit lung Annexin I matched the aminoacid sequence between Ser-37 and Leu-51 of human Annexin I whose entireamino acid sequence had been deduced from cDNA as described in theliterature. Among the 15 amino acids, Thr-41 in human Annexin I sequencewas replaced with Phe in the 33 kDa(BP) protein and Asp-47 in humanAnnexin I sequence could not be determined for the 33 kDa(BP) protein.

The results of Western blot also showed that the immunoreacted Annexin Iin human BAL fluid had the same apparent molecular weight as rabbit lungAnnexin I. The molecular weight of human Annexin I calculated from aminoacid sequence deduced from cDNA is 38712.16. Recently, the rabbitAnnexin I cDNA was cloned and sequenced; the deduced protein sequencehas 346 amino acids with a calculated molecular weight of 38831.28,similar to the Annexin I of human, rat, mouse and guinea pig. Theapparent molecular weight of rabbit lung Annexin I was 36 kDa asvigorously examined by SDS gel. The observed molecular weights of humanAnnexin I also have been reported to be around 35-37 kDa. The differencein molecular weights between the calculated and experimentallydetermined might be the result of protein charge effects on proteinmigration on SDS gel due to some post-translational modifications. To beconsistent with the observed molecular weight, lung Annexin I isreferred to as 36 kDa(PLBP) protein since Annexin I was analyzed mostlyby SDS-PAGE and Western blot.

III. Inhibition of Endotoxin and Cytotoxicity by Annexin I and AnnexinVIII i. In vitro Study of Anti-endotoxin and Anti-cytotoxicityActivities of Annexin I and Annexin VIII

I have discovered that both Annexin I and Annexin VIII effectivelyinhibited endotoxin stimulation on macrophage release of cytotoxinsubstances. Also, Annexin I and Annexin VIII inhibited killing of cellsby cytotoxin substances released by activated macrophages. Overall,Annexin I and Annexin VIII prevented endotoxin toxicity.

In the same in vitro cytotoxic assay in which it was demonstrated thatboth Annexin I and the Annexin VIII protein prevented the killing ofcells by the 33 kDa(BP) breakdown product of Annexin I.

ii. Annexin I and Annexin VIII Inhibit Cytotoxicity of BAL Fluid from CFPatients and BPD Patients

I have found that the BAL fluid from either CF patients or BPD patientskilled cells to a certain extent. These BAL fluid samples contained the33 kDa(BP) but had no Annexin I. I also discovered that when Annexin Ior Annexin VIII was added to the cell culture medium, these proteinsprotected cells from killing by the BAL fluid of CF or BPD patients.

iii. In vivo Study of Annexin I Against Endotoxin

In this experiment, 6 New Zealand white rabbits (40-days old) wereanesthetized with Ketamine followed by injection of 2 ml of saline intothe tracheas of two control rabbits, 2 ml of saline containing 0.2 mgLPS into two Endotoxin group rabbits, or 2 ml of saline containing 0.2mg LPS and 0.2 mg purified rabbit lung Annexin I into twoEndotoxin+Annexin I group rabbits. Anal temperature of each rabbit wasmeasured every half or one hour after injection.

A tracheal injection of LPS into rabbits induced response within onehour. The animals had body temperatures 2-3° F. higher than the controlswithin 5 hours. The body temperatures of the rabbits returned to normallevels 6 and 7 hours after injection. The injection of a mixture ofLPS-Annexin I-calcium delayed the effects of endotoxin more than 2 hoursand reduced the degree of response. The rabbits that received Annexin Ieventually developed fever 4 hours later after injection. This isprobably due to only one level of Annexin I being tested versus a largedose of endotoxin and the removal or degradation of Annexin I in theairway and the residual endotoxin causing infection. The differencebetween the Control and the average of the Endotoxin andEndotoxin+Annexin I groups for all times 3 hours and after was −1.97degrees F. (p-value<0.001) while the difference for these times betweenthe Endotoxin and Endotoxin+Annexin was 0.85 degrees F. (p-value 0.014).This indicates that, after a brief incubation period, theEndotoxin+Annexin I group had a significantly lower average temperaturethan the Endotoxin group.

All the rabbits used in the in vivo study (trachea injection ofendotoxin, Annexin I and endotoxin, or saline) were normal rabbits andthey had endogenous Annexin I. This shows that introducing additionalexogenous Annexin I into the airway of even healthy rabbits can inhibitendotoxin toxicity. The quantity of Annexin I in the airway is importantin protecting against endotoxin. Therefore, introducing exogenousAnnexin I and/or Annexin VIII into an animal's airway will enhance theanimal's defense mechanism.

The Annexin I anti-endotoxin activity is probably due to Annexin Ibinding to the lipid-A moiety of LPS so the LPS can no longer bind tothe host cell membrane to trigger cellular reactions that releaseinfectious mediators. Annexin I may also bind to the epithelial cellsurfaces to prevent LPS from anchoring on the cell membrane to initiatecellular reactions. All the results indicated that Annexin I and AnnexinVIII can effectively inactivate endotoxins. Moreover, since thesecompounds are natural products of the lung their administration into theairways of animals, including humans, has minimal or no side effects.

IV. Uses for the Annexin I Breakdown Product, 33 kDa(BP)

33 kDa(BP) is an endogenous cytotoxic substance which can be used incell culture and animal models to study cytotoxicity and apoptosis inlaboratories. Since endogenous 33 kDa(BP) is not easy to obtain, thecommercial production of 33 kDa(BP) will be useful. 33 kDa(BP) can bereadily made in bacteria by the recombinant DNA techniques. Thepost-translational modification of the protein is not a concern insynthesizing 33 kDa(BP) in bacteria since Annexin I charge modificationoccurs at the N-terminus which is depleted in 33 kDa(BP) anyway.

The presence of 33 kDa(BP) in the diseased or inflamed lung appears tobe a critical endogenous apoptosis factor that causes epithelial celldeath and lung injury. Thus, the Annexin I/33 kDa(BP) ratio in BAL fluidcan be used as diagnostic tool to predict lung injury. I have discoveredthat Annexin I and/or 33 kDa(PLBP) can effectively inhibit the cytotoxicactivity of the Annexin I breakdown product 33 kDa(BP).

The 33 kDa(BP) also can be useful in the development of new drugs toinhibit 33 kDa(BP) cytotoxic activity. Inhibition of 33 kDa(BP)cytotoxicity can lower a patient's susceptibility to lung inflammationand enhance the patient's recovery rate. The discovery of 33 kDa(BP)therefor permits specific inhibitors to be developed to inhibitcytotoxicity in the airways of patients with lung diseases and to lowera patient's susceptibility to lung injury. I have discovered thatAnnexin I and/or Annexin VIII can effectively inhibit the cytotoxicactivity of the Annexin I breakdown product 33 kDa (BP).

33 kDa(BP) and the ratio of 33 kDa(BP)/Annexin I in BAL fluid also canbe used in a diagnostic kit to diagnose or predict lung injury by usinga specific antibody to 33 kDa(BP) to detect the presence of 33 kDa(BP).

V. Analysis of Annexin I in Human Amniotic Fluid (AF)

I also have discovered that the presence of Annexin I and 33 kDa(BP) inamniotic fluid can be useful in diagnosing high risk pregnancies.

In four AF specimens from four patients with high risk pregnancies,Annexin I and 33 kDa(BP) were detected in two of the AF specimens,whereas two AF specimens contained no Annexin I but only 33 kDa(BP). Itis likely that these latter amniotic fluid specimens containedproteolytic enzymes which hydrolyzed Annexin I to yield the 33 kDa(BP).In addition, preliminary data showed that the breakdown of Annexin I inamniotic fluid from patients with high risk pregnancy was similar tothat in the BAL fluid from patients with lung inflammation. Elastaseinhibitor (PMSF) prevented Annexin I degradation in the presence ofelastase.

In addition, to using the analysis of the amount of Annexin I/33 kDa(BP)in amniotic fluid as a means to predict premature delivery, theadministration of Annexin I and/or Annexin VIII into the amniotic fluidwill help to prevent the infections, effects of cytotoxins andprostaglandins which are the major causes of premature delivery. Themechanism of the infection in amniotic fluid is similar to that in theairway.

VI. Inhibition of PLA₂ and Suppression of PLA₂ Stimulation

PLA₂ plays a pivotal role in the pathway which leads to the productionof inflammatory lipid mediators. I have found that there are PLA₂stimulators that can be present in the BAL fluids of patients with lungdisease which can stimulate the activity of PLA₂. I have also discoveredthat Annexin I, Annexin VIII or mixtures thereof can inhibit both PLA₂activity and suppress the activity of PLA₂ stimulators in the BAL fluidsof patients with lung disease. The PLA₂ stimulator activity in the BALfluid was measured by the augmentation of PLA₂ activity on phospholipidhydrolysis. Annexin I or Annexin VIII was introduced into BAL fluids andfound to inhibit the activities of both PLA₂ and the PLA₂ stimulator.The BAL fluids used in this study were from patients with cysticfibrosis (CF) or bronchopulmonary dysplasia (BPD) and were obtained fromthe University of Wisconsin Medical School. The PLA₂ and phospholipidsemployed were commercially available. The Annexins I and VIII wereisolated from rabbit lungs by the inventor.

I first looked at bronchoalveolar lavage fluids (BAL) of patients withcystic fibrosis, and was surprised to find no increase in PLA₂ activity.However, when I added BAL from a cystic fibrosis patient to apreparation of porcine pancreatic PLA₂ in a test tube, I saw a largeincrease in activity of PLA₂. This stimulatory activity was notdestroyed by incubating the BAL for one hour at 37° C., nor was itdestroyed by boiling for 5 minutes. No such stimulator activity waspresent in the BAL from normal volunteers. The following assay for PLA₂activity was used.

Phospholipase A₂ Assay

A reaction mixture in 0.1 ml of 0.01 M Tris-HCl buffer (pH 7.4)contained 10 MM CaCl₂, 5 nmol unilamellar liposomes made of dioleoylphosphatidylcholine (PC) and phosphatidylglycerol (PG) (50%-50%, by wt)labeled with L-α-[1-¹⁴C]dioleoyl PC, 25-100 μg of CF BAL fluid proteins,10 μg of either rabbit lung Annexin I or Annexin VIII, and 0.5 μg ofporcine pancreatic phospholipase A₂. In the control reaction mixture,either CF BAL fluid, Annexin, or both CF BAL fluid and Annexin wereomitted. The reaction was carried out at room temperature for 20 secondsand stopped by adding 2 ml of chloroform:methanol (1:2 vol) followed byaddition of 0.4 ml water and 10 μl of egg PC (20 nmol) which was used ascarrier. The mixture was stirred, lipids were extracted by addingadditional 0.6 ml water and 0.6 ml chloroform. PC was isolated by themethods of silica gel thin-layer chromatography (TLC). Lipids on the TLCplate were visualized by exposure of the TLC plate in an iodine tank.The PC spot on the plate was scrapped into a scintillation vial,radioactivity determined by a scintillation counter. Phospholipase A₂activity was expressed as either decrease in PC radioactivity orincrease in fatty acid or lysoPC radioactivity as described previously(Tsao et al., BBA 1081: 141-150, 1991). In additional studies CF BALfluids were incubated in boiling water for 4 minutes followed bycentrifugation at 10,000 rpm for 10 minutes prior to its addition to thephospholipase A₂ reaction mixture.

The above procedure was used to identify a novel PLA₂ stimulating factor(PLA₂ stimulator) in bronchoalveolar lavage (BAL) fluids from patientswith cystic fibrosis (CF). The PLA2 stimulator activity was measured bythe level of increase in porcine pancreatic PLA₂ activity on hydrolysisof ¹⁴C-labeled dioleoyl phosphatidylcholine (PC) in the presence of CFBAL fluid. The CF BAL fluid stimulated PLA₂ activity in a BAL fluiddose-dependent manner; the stimulation reached 2-fold by 50 μg of totalproteins in the fluid and was maintained at optimal levels with the BALfluids containing 100 μg of proteins. The CF BAL fluid itself had nomeasurable PLA₂ activity. Bronchoalveolar lavage fluids from normalvolunteers had no stimulation on PLA₂ activity. The PLA₂ stimulationactivity in the CF BAL fluid was stable after the fluid was heated inboiling water for 4 min, and the activity was recovered in the 10,000rpm supernatant of the heated BAL fluid. The PLA₂ and PLA₂ stimulatoractivities were markedly inhibited by Annexins I and Annexin VIIIisolated from rabbit lung. The results show that BAL fluids frompatients with CF contained PLA₂ stimulator(s), and both the PLA₂ andPLA₂ stimulator activities could be inhibited by lung Annexins I andVIII. These results support the conclusion that a PLA₂ stimulator is acritical factor involved in inducing inflammation and that Annexins Iand VIII may have a role in suppressing the PLA₂-mediated inflammation.

The discovery that both PLA₂ activity can be inhibited and the PLA₂stimulatory activity of simulators can be suppressed by adding Annexin Ior Annexin VIII to a body fluid, such as lung fluid, is an importantone. Annexins I and VIII are native lung proteins present in the airwaythat play a role in host defense.

In further experiments, I found that the BAL from patients withbronchopulmonary dysplasia (BPD) also contained PLA₂ simulators for theactivity of PLA₂ and that the breakdown products of Annexin I, whichwere in themselves cytotoxic, were ineffective at inhibiting PLA₂ orPLA₂ stimulator activity. I previously found that the addition ofAnnexins I and VIII prevented a cytotoxicity seen from the BAL from CFand BPD patients. This evidence supports the conclusion that bringingAnnexin I, Annexin VIII or a mixture thereof into contact with lungfluid of a patient can inhibit both PLA₂ activity and any PLA₂stimulator activity and thereby interrupt the pathway involving PLA₂that leads to inflammation.

The surprising discovery of the PLA₂ stimulator activity in the BAL ofpatients with lung disease provides a better understanding of themechanisms of PLA₂-mediated inflammation making it possible to developnew drugs to inhibit this activity.

The embodiment of the method of the present invention by which acandidate anti-inflammatory agent can be tested to determine if it willinhibit the stimulation of the activity of PLA₂ by natural PLA₂simulators known to be present in a body fluid comprises adding thecandidate agent to the body fluid and after a reasonable time (e.g., 20seconds) testing the body fluid to see if any PLA₂ activity has beenstimulated. The results obtained may be compared to the results obtainedin a control sample of the body fluid subjected to the same conditions.

As previously stated, the preferred pharmaceutical compositions, inaddition to Annexin I, Annexin VIII, or mixtures thereof, will contain asource of calcium ions. When intended for instillation into the lungs ofan animal, they also will contain a surfactant or surface active agent.

The Annexin I and Annexin VIII are natural proteins in the airway.Therefore, the side effects and toxicities of these proteins areminimal. Both human Annexin I and Annexin VIII can be synthesized orproduced by genetic engineering techniques.

The preferred source of calcium ions is a soluble calcium salt, such ascalcium chloride. The amount of calcium ions in the compositions willdepend upon the amount of active ingredient. The calcium ionconcentration can range from zero where enough endogenous calcium ispresent to about 1 mM or more.

A preferred surfactant would be one that is known to be beneficial inthe treatment of lung disease, such as that sold under the trademarkSURVANTA by Ross Laboratories. Suitable surface active agents includeboth non-fluorinated surfactants and fluorinated surfactants known inthe art and disclosed, for example, in British Patent Nos. 837465 and994734 and U.S. Pat. No. 4,352,789.

Examples of other surfactants include:

Sorbitan trioleate,

Sorbitan mono-oleate,

Sorbitan monolaurate,

Polyoxyethylene (20) sorbitan monolaurate,

Polyoxyethylene (20) sorbitan mono-oleate,

Lecithins derived from natural sources

Oleyl polyoxyethylene ether,

Stearyl polyoxyethylene,

Lauryl polyoxyethylene ether, and

Oleyl polyoxyethylene ether.

The surfactants or surface active agents are generally present inamounts not exceeding 5 percent by weight of the total formulation. Theywill usually be present in the weight ratio 1:100 to 10:1 surface activeagent: active ingredient, but the surface active agent may exceed thisweight ratio in cases where the active ingredient concentration in theformulation is very low.

The particle size of the active ingredients should desirably be nogreater than 100 microns diameter. Preferably, the particle size of afinely-divided solid powder should for physiological reasons be lessthan 25 microns and preferably less than about 10 microns in diameter.The particle size for inhalation therapy should preferably be in therange 0.5 to 10 microns.

The concentration of the active ingredient depends upon the desireddosage, but it is generally in the range 0.01 to 5% by weight. Thedosage will usually be selected to bring the levels of the Annexin I orAnnexin VIII at least up to the levels of those polypeptides found inthe lung or amniotic fluid of normal animals. It should be noted,however, that the dosage can be adjusted to any level which is toleratedwithout substantial adverse effect by the patient. Thus, dosages of from0.05 μg/kg of the animal's body weight up to 500 mg/kg or higher couldbe used if such high levels are not toxic and produce the desirableresult in the patient. The availability of an animal model for cysticfibrosis, mice homozygous for a disrupted CFTR gene, allows for thetesting of compositions containing the active ingredients in animalswithout undue experimentation.

A representative composition for instillation into the lungs of animalwould contain in 1 ml. about 0.02 mg of Annexin I, Annexin VIII, or amixture thereof; 0.5 mg of calcium (5 mM) and 0.4 mg of surfactantphospholipids. It could also contain other diluents and ingredients andit would be packaged in an aerosol form not requiring a CFC propellant.A composition for addition to amniotic fluid would not contain thesurfactant.

From the foregoing, it will be apparent to those skilled in the art thatmy discovery that the addition of Annexin I, Annexin VIII or mixturesthereof to body fluids will inhibit PLA₂ activity and PLA₂ stimulatoractivity has wide and broad clinical applications for the treatment ofinflammation in a wide variety of patients, such as children and adultswith cystic fibrosis, HIV/AIDS immune suppressed patients, neutropenic,post-operative, bed ridden and chronic obstructive pulmonary diseasepatients, as well as, infants with bronchopulmonary dysplasia andpatients with septic shock or patients who have high risk pregnancies.

It will be apparent to those skilled in the art that a number ofmodifications and changes can be made without departing from the scopeof the invention. Therefore, it is intended that the invention belimited only by the claims.

What is claimed is:
 1. A method of inhibiting the activity ofphospholipase A₂ (PLA₂) and suppressing the activity of PLA₂ stimulatorsin the airway of an animal which comprises contacting said airway with acomposition comprising a member selected from the group consisting ofAnnexin I, Annexin VIII and mixtures thereof, in an amount which willinhibit PLA₂ and suppress the activity of PLA₂ stimulators.
 2. Themethod of claim 1, wherein the composition contains a surfactant.
 3. Themethod of claim 1, wherein the composition is an aerosol or spray.